Identification of a roseoflavin exporter in the roseoflavin producer Streptomyces davaonensis

达文链霉菌在生长的固定阶段产生抗生素玫瑰黄素。玫瑰黄素是一种结构核黄素（维生素B2）类似物，具有抗代谢作用。Mack小组能够证明玫瑰黄素对FMN核开关和黄素蛋白产生负面影响，因此抗生素具有多个细胞靶标。Mack小组还开展了有关玫瑰黄素生物合成途径的基础工作。目前一个重要的悬而未决的问题是玫瑰黄素如何从生产者细胞出口。我们的初步实验清楚地表明，一种未知的转运体负责玫瑰黄素的输出，因为在生产者细胞的培养上清中发现了玫瑰黄素。这种输出蛋白必须具有高度特异性，以避免必需核黄素的输出。玫瑰黄素的出口对生产者很重要，否则该化合物将无法在自然环境中抑制竞争微生物的生长。本拨款申请的目的是鉴定玫瑰黄素出口的S. davaonensis。五个不同的工作包描述了应该导致识别玫瑰黄素出口商的实验。目前尚不清楚是单一蛋白质还是多种蛋白质形成了这种独特的出口蛋白。我们计划在不同的细菌背景下进行系统的缺失实验和共表达实验来鉴定玫瑰黄素出口菌。如果这些实验失败，我们计划进行蛋白质-蛋白质相互作用实验。因此，假设玫瑰黄素生物合成的最后一种酶RosA与其反应产物玫瑰黄素结合非常紧密，与膜结合的出口酶相互作用。也有可能玫瑰酸与一种结合蛋白相互作用，然后将玫瑰黄素带到难以捉摸的出口者那里。利用蛋白-蛋白相互作用研究可以鉴定玫瑰黄素的结合伙伴，这可能有助于鉴定玫瑰黄素输出系统的组分。原则上，细菌的摄取系统——尤其是核黄素的摄取系统——已经得到了很好的描述。相比之下，只有极少数出口商是已知的，这使得目前的赠款申请特别有趣。

The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin in the stationary phase of growth. Roseoflavin is a structural riboflavin (vitamin B2) analog and acts as an antimetabolite. The Mack group was able to show that roseoflavin negatively affects FMN riboswitches as well as flavoproteins and thus the antibiotic has multiple cellular targets. The Mack group also has carried out fundamental work with regard to the biosynthetic pathway of roseoflavin. One important open question is at present how roseoflavin is exported from the producer cell. Our preliminary experiments clearly show that a yet unknown transporter is responsible for roseoflavin export as roseoflavin is found in the supernatant of cultures of the producer cell. Such an exporter protein must be highly specific to avoid export of essential riboflavin. Export of roseoflavin is important for the producer, otherwise the compound would not be able to inhibit growth of competing microorganisms in a natural environment. The objective of the present grant application is the identification of the roseoflavin exporter in S. davaonensis.Five different work packages describe experiments which should lead to the identification of the roseoflavin exporter. It is unclear at present whether a single protein or whether multiple proteins form this unique exporter. We plan to carry out systematic deletion experiments and co-expression experiments in different bacterial backgrounds to identify the roseoflavin exporter. In case these experiments fail we plan to carry out protein-protein interaction experiments. The hypothesis thereby is that the last enzyme of roseoflavin biosynthesis, RosA, which binds its reaction product roseoflavin very tightly, interacts with a membrane bound exporter. It is also possible that RosA interacts with a binding protein which then brings roseoflavin to the elusive exporter. Employing protein-protein interaction studies binding partners of RosA can be identified, which may help to identify components of the roseoflavin export system. In principle uptake systems – especially for riboflavin - are well described in bacteria. In contrast, only very few exporters are known which makes the present grant application especially interesting.